- [Presenter] This program is presented by University of California Television. Like what you learn? Visit our website or follow
us on Facebook and Twitter to keep up with the latest UCTV programs. (upbeat music) - Good afternoon, everyone. I'm Mary Comerio, professor
of the Graduate School in Architecture and chair of the Hitchcock Professorship Committee. We are pleased, along
with the Graduate Council, to present Carol Greider,
this year's speaker in the Charles M. and Martha
Hitchcock lectur
e series. As a condition of this request, we are obligated and happy to tell you how the endowment came to UC Berkeley. It is a story that
exemplifies, in many ways, the campus linked to the
history of California and to the Bay Area. Dr. Charles Hitchcock, a
physician for the army, came to San Francisco
during the gold rush, where he opened a
thriving private practice. In 1885, Charles established a professorship here at Berkeley as an expression of his
long-held interest in education. His daugh
ter, Lily Hitchcock Coit, still treasured in San Francisco for her colorful personality
as well as her generosity, greatly expanded her
father's original gift, to establish a professorship
at UC Berkeley, making it possible for us to
present a series of lectures. The Hitchcock fund has become one of the most cherished endowments of the University of California, recognizing the highest distinction of scholarly thought and achievement. Thank you, Lily and Charles. And now, a few words about Carol
Greider. Carol Greider is internationally renowned for her important contributions to the field of molecular biology. She was awarded the 2009 Nobel Prize for physiology of medicine, along with Elizabeth
Blackburn and Jack Szostak, for their discovery that genetic sequences known as telomeres are protected from progressive shortening
by the enzyme telomerase. Greider discovered telomerase in 1984, while working with Elizabeth Bradford at the University of California Berkeley. Telomeres are essen
tial to chromosome maintenance and stability. In the absence of telomerase, telomeres shorten
progressively as cells divide, eventually leading to cell death or cellular senescence. Greider's understanding,
Greider's breakthrough discovery of telomerase enzyme that allowed a mechanistic understanding
of cellular reproduction, opening new avenues of
research into cancer and age-related diseases. Greider received her BA in biology from the University of California, Santa Barbara, in 1983, her
PhD
in molecular biology at the University of
California, Berkeley, in 1987. She was a Cold Spring Harbor fellow and later held a faculty position at the Cold Spring Harbor Laboratory in Long Island, New York. In 1997, she joined the faculty of the Department of
Molecular Biology and Genetics at Johns Hopkins University where she is currently the
Daniel Nathans professor and director of molecular
biology and genetics, as well as professor of oncology in the John Hopkins
University School of Medicine
. Greider also directs her
own research laboratory at the University which conducts research into telomere function and structure. In recognition of her
groundbreaking research, Greider has been named a fellow by the American Academy of Microbiology, the American Association for
the Advancement of Science, the American Academy of Arts and Sciences, and as a member of the
National Academy of Sciences and the Institute of Medicine. In addition to her Nobel Prize
for physiology of medicine, she has
received a number of other awards in the last 10 years, including the Pearl
Meister Greengard Prize, the Paul Ehrlich, Ludwig
Darmstaedter Prize, the Kathryn Burke and Judd Award, the Richard Lounsbery Award,
the Dickson Prize in Medicine, the Wiley Prize in Biomedical Sciences and the Louisa Gross Horowitz Prize. An amazing list. Greider has also published over 100 articles and book chapters. Please join me in welcoming
Carol Greider back to Berkeley. (audience applauding) - Well, it's really
a pleasure to be here. I've been looking forward
to this lecture series for a number of months now. It's always a pleasure to
come back here to Berkeley and to be here in the I-House. Not only did I get my PhD here at Berkeley but both my mother and my father received their PhDs here at Berkeley so it really is like a homecoming. So thank you very much
for this invitation. So what I would like to do today, is to give you a sense of the background of why we thought telomeres and telomerase were i
nteresting and
take you on a little bit of a journey from the initial discovery of the function of telomeres, through how, now, we understand they play a role in age-related disease So the theme of this
lecture is going to be that curiosity-driven
research can establish fundamental knowledge that
fuels practical applications. So we'll start at the beginning or perhaps we'll start at the end because telomeres are at both the beginning and the ends of chromosomes. So if you have, within a cell, th
e nucleus and in the nucleus
are all of the chromosomes and this is a blow-up of
one of those chromosomes and so most of what is studied in the field of molecular biology occurs along the length
of the chromosomes, where all of the genes and
the control elements lie but we'll be focusing today on this region at the end which is called the telomere. Telomeres provide two really important functions for the cell. First of all, they have to
protect the chromosome ends. They protect the chromosome en
ds from anything that would chew away, a nucleus that may chew
a way from the end. They protect the ends from
recombination with other ends and they protect the ends
from fusion with other ends. So this is a really essential function for chromosome maintenance. The other thing that telomeres have to do, is to allow the maintenance
of chromosome length and we'll be going through
why that is very important throughout most of the rest of the talk. So telomeres were first
functionally defined by H.J
. Muller and by Barbara McClintock back in the late 30s and early 40s. So, Muller, working in Drosophila, showed that telomeres were essential to protect chromosome ends and McClintock was working in the plant, maize or corn and also came to the same conclusion, that the telomeres were necessary for protecting the chromosome ends. So this was a very early discovery and it wasn't possible
to really understand, in molecular detail, what a telomere was or how carried out its function
until it was c
haracterized at the very molecular level. And so that occurred in the 1970s with this organism here,
Tetrahymena thermophila. So Tetrahymena is a single-celled, ciliated protozoum. If you went to the creek, you'd be able to take some pond water and see ciliates swimming around. And the Tetrahymena is very unique because a single cell
contains 40,000 chromosomes. So this was the organism of choice to try and identify what
is the molecular basis for the function of these telomeres. And so in 1978,
Elizabeth Blackburn, working together with Joe Gall, were able to define the molecular sequence, the DNA sequence that's at the very end of the chromosome. And what they found is that
there was a very simple, repeated DNA sequence. So the DNA has normally four
different building blocks, T, G, C, and A. In this case, it was simple repeats of two Ts followed by four Gs and then hundreds of those repeats, on the ends of the chromosomes. So this was a very
important initial finding because it then
allowed
additional experiments to be carried out to understand
the function of telomeres. So here's Joe gall and Liz Blackburn. I'm going to introduce
a number of the people along the way because one of the themes in science is, of course,
it's not just one person making a discovery but many people building on what others have done. So I'll try and give you a flavor of some of the people that
have done the experiments that I'm talking about, as I go along. So the first identification
of the DNA
sequences of telomeres, as I said, was in this ciliated
protozoan tetrahymena. And the telomere sequence
was identified as T2, G4, repeats of this sequence. And then subsequent work by a number of other laboratories was able to show that very similar, tandemly repeated, simple GT-rich sequences were present in a variety of other organisms. So for instance, in fungi, there's a model organism,
Saccharomyces cerevisiae. And here, the sequence was T, followed by either one,
two, or three G residues.
And then I'll also be talking about the human telomeric sequence. And in all vertebrates, including humans, the sequence is TTAGGG,
repeated many times. So this theme, it turns out, repeats itself throughout
various organisms and this is one thing that's very nice about working on something
that is very fundamental to biology and that is
that the same themes occur throughout many different organisms. So the telomeric DNA contains these tandem repeats of simple sequences. And of course, most of
the DNA in a cell is double-stranded, so you have both the TG-rich sequence, as well as a CA-rich sequence. Although there is a region at the very end of the chromosome in which
it's single-stranded. But in order to carry out
this function of telomeres, you require not just
the DNA but of course, there have to be proteins
that bind to that DNA, that actually carry out the function of the protecting the chromosome. and in mammalian cells, this is a complex of proteins called the Sheltering Comple
x, which protect the chromosome ends and so there are proteins that bind along the length of the
double strand of the DNA, proteins that to those proteins, and the proteins that also protect this single strand region. And it's together, this complex of the proteins in the DNA, that carry out the telomere function that
we'll be discussing today. So it turns out that it
was first hypothesized by Jim Watson and by others, that if you look at the mechanism by which chromosomes are copied, every time
a cell has to divide, that it was predicted that the chromosomes should shorten from their ends
every time the cell divides. And this is because of
the molecular mechanism that replicates the entire
length of the chromosome. So what one would predict
is that every time a cell divides, the
telomeres should shorten from their ends. And so you would have a loss
of these telomeric repeats. However, it turns out that
the way most organisms that have these linear chromosomes overcome this problem in
replication is through this enzyme telomerase and that's what we were able
to show from Tetrahymena. So you may have some
telomere shortening here but then the telomerase
will add these repeats back on to the ends of chromosomes so that chromosomes
aren't a unique length, rather there's an equilibrium. There's always some shortening
and then some lengthening and some shortening and some lengthening and that way, the average length of the average telomere
is maintained over time. So this is a rat
her baroque mechanism that doesn't really avoid
the shortening problem but rather, overcomes it. So we've been interested in this telomerase enzyme
for a number of years. And of course, we don't
usually look at it, as this kind of a little cartoon, we're interested a little bit more in the details of the mechanism. But I won't really have a chance to get into many of
those details here today but I'll just show you
some of the components that go into this so telomerase, so that I can tell you a l
ittle bit about how we study its role. So the telomerase is a
really remarkable enzyme. It actually contains protein components as well as an essential RNA component. So this is unusual for an enzyme. So this stick figure here
is meant to represent the RNA component of telomerase. And the RNA component of telomerase actually has a region which can base pair with the telomere to be able to add these telomeric repeats onto
the ends of the chromosome. And I'm introducing
this, not so much today, to
tell you about how
telomerase does this, which is really a fascinating question, but rather to introduce
you to the components of telomerase that I'll be
discussing throughout the talk. So this protein here that carries out a telomerase function, we call a TERT, for telomerase reverse transcriptase. And so this carries out the catalytic function of telomerase. And this RNA component, we imaginatively call telomerase RNA, abbreviated, TR. So you'll be hearing
about these components throughout th
e rest of the talk. So having discovered that the way that telomeres are maintained, rather than having them shorten every time the cells divide, rather to be maintained by telomerase, we were
very curious to know what would happen if you
don't have telomeres? What would be the consequence of not being able to
elongate the telomeres? And so that led us, and
a number of other labs, to two really fundamental
consequences of telomerase. So the question is why
does telomerase matter? And it turns ou
t that it's required for all cells that have
to divide many times. So I'll be telling you
a little bit of a story where telomerase is required
for the growth of cancer cells. So if you imagine that
you have a particular set of cells here. One of those cells undergoes
some kind of a genetic change, which allows it to divide more times than it normally should. So that's this red cell here. And that red cell then will go and divide many more times relative to
the other cells around it. And it turns
out, in order for that cell to continue dividing and form that tumor, it has to solve this telomere problem, that is the problem with
the telomere shortening every time the cells divide. So these cancer cells need have telomerase in order to maintain their telomeres to go on and continue dividing. So I'll tell you a little bit about that. Telomerase is also essential for a number of other cells in our body which have to also divide many times and these would be stem cells
or tissue specific ste
m cells, where you have a cell that makes a copy of itself but then also has to make what are called pluripotent stem cells, these are our cells that will go on and make the final tissue. So this, again, are
cells that have to divide many times in order to replenish the other cells in the body. So these are the two areas that the telomerase clearly
plays an important role. And we'll be talking,
toward the end of the talk, about how this role here plays a role in age-related
degenerative disease.
So we were very curious
about the role of telomerase and what might happen if
you didn't have telomerase, so we set out to generate a mouse which doesn't have this activity. So instead of being able
to maintain the telomeres by adding repeats onto the end, we generated a knockout so
it had no telomerase at all. And then we wanted to study what happens to that mouse when the
telomeres get to be short. So the way that we generated this mouse that doesn't have telomerase, was we had identified, as
I told you, the telomerase RNA, that's
abbreviated here, TR. The M stands for the mouse, so it's the mouse version
of the telomerase RNA. And we generated mice
because mice are diploid. Of course, they have two
copies of chromosomes. One from their mom and one from their dad. So we generated these mice
which are heterozygous, in which, they have one normal copy of the telomerase RNA
and then the other copy is just missing the telomerase RNA. So it's a minus. So it has one copy instead of two co
pies. When you then take two such mice and you breed them together, as Mendel told us from genetics, what you would get by breeding
these two mice together, is you get mice that have two copies of the telomerase RNA, mice
that are heterozygotes, or have, like their parents, one normal copy and one missing copy or you get the mice
that are completely null for telomerase and have
no enzyme whatsoever. So we generated these mice and remarkably, the mice were perfectly fine. There was nothing wrong
with them at all and so we were curious what might happen over time with these mice. So we designated these mice as the G1, that is the first generation
that lack telomerase. So these G1 mice were
then bred to each other and of course, you'd breed the G1 mice, so these are null. You get the G2 knockout mice. You breed those mice together, you get G3. Et cetera, G4, G5, and G6. And I'll be showing you
that in these G6 animals, we can't get a later generation because one of the things that happens
, is these mice become infertile. There are a number of other consequences of the loss of telomerase but we don't get future generations. So as these mice are breeding, what we found is that the telomeres, with each generation of mouse, were getting shorter and shorter. So the cartoon version
of this is shown here. We breed the G1 mice and we get the G2. We take these two G2
mice and now the G3 mice have shorter telomeres
than their parents did. And then you breed the
G3 mice to each other and t
he G4 mice have
even shorter telomeres. So with progressive generations, there was progressive telomere shortening and the way that we actually look at this in the laboratory is a
very nice visual assay that was developed by Peter Lansdorp. And what this is, is a way to look at all of the chromosomes in the mice. So these are metaphase spreads. So you take the the cells
when they're about to divide and you can visualize the chromosomes which are shown here in blue, with a stain that's staining t
he DNA and then the telomeres are visualized with a fluorescent probe that recognizes the telomere and the
intensity of this spot, that is how many probes
are hybridizing there, is proportional to how
long the telomere is. So the brighter the spot, that means you have a longer telomere. And this is a very nice assay because you can actually
see what's happening to the chromosomes. So this is how we were able
to look at telomere length. And what we found when
you breed these mice down in this nic
e line here is if you look at the intensity of each one
of those red dots from before versus the number of dots
that have that intensity, you have this nice distribution and this distribution is the normal way that telomeres are maintained and they're maintained
about this equilibrium because there's some
shortening and lengthening and shortening and lengthening. However, with each generation, now, this is the G2, the G4, and the G6, you can see that there's
progressively shorter and shorter tel
omeres with each generation that these mice are bred. One of the nice things
about this visual assay, where you're actually
looking at the chromosomes, is that we could show that not only are we losing telomeric
sequence with each generation, but we're actually losing
telomeric function. And I'll be telling you, in just a minute, that we see effects that is phenotypes in
these later generation mice that we don't see in the
the first generation mice, indicating that the short telomeres are what's
causing effects in these mice. So when we actually look at the different generations of mice, here is the wild-type mouse chromosomes that I showed you, with these red dots on the ends of the chromosomes. And you can see that
these mouse chromosomes look like these little boomerangs, they look like little Vs because the centromere is all the way along one end of the chromosome. So they're attached here and then the two long arms are going off here. So you see this nice spread
of mouse chromosom
es. In the second generation,
you also see that, and also the fourth and in the sixth. But what was remarkable is that in these very later generations, what you see here is what
looks like two chromosomes that have now fused through their ends. Instead of being little boomerangs, they're two that are
attached to each other and what that indicates is that not only have we lost telomere sequence, but we've lost telomere function. Because I told you that what telomeres do is to protect the chromoso
me ends and it protects them from
these kinds of recombination. So we can actually see this loss of telomere function visually. So the mice, once they get to be in the
fifth and sixth generation, it turns out, have a number
of problems with them. So I mentioned, in the testes, that these mice become infertile. So there's actually a cell
death of the germ cells. In the intestine, the
normal intestinal cells which allow cell turnover
isn't occurring properly, so you have this atrophy. In the blood
, there's bone marrow failure. So the blood is one cell type where you have to have
cells continually renewing because blood cells are dying every day. In the skin, there's
decreased wound healing and there was premature graying of these late generation mice. And it was remarkable that
all of these phenotypes seen in these mice were seen in the fifth and sixth generation mice. Okay, they were seen when
the telomeres were short but not in the first generation mice when telomerase was absent. So w
hat that tells us is
that it takes some time for the telomeres to get short and it must be the short telomeres that are causing these effects, okay? So it's not the absence
of the telomerase enzyme because if it were the
absence of telomerase, you would see that in the
very first generation. The fact that we have to
wait for six generations means it must be the short telomeres that are causing these effects. So I'm going to tell you now about work from a student
who was able to show that it was
actually
the shortest telomeres that that caused this effect. And this was Mike Hemann. And Mike did a series of experiments, crossing mice with long telomeres and mice with short telomeres and what he was able to to show is that it's actually
the shortest telomere that leads to either
cell death or senescence. So just like the different
generations of mice, the mice with the long
telomeres, there's no effect. As those cells are dividing now, the telomeres become short
and the shortest telomere
in the cell is actually recognized and it tells the cell that
there is damage going on and what happens is the
cells either undergo cell death or cellular senescence. So cell death, you actually
get the absence of the cells and cellular senescence is a phenomenon where the cells just stop dividing and they won't divide any further but they're still staying around. So it's clearly the short telomeres that are causing this effect. So we were interested,
then, in the consequence of this short telom
eres
causing cell death. And so I'll tell you one short story where we were interested
in how this may play a role in cancer. So we were interested in a
particular kind of cancer and that's a cancer that
causes B-cell lymphoma. So this is a leukemia type cancer. And model mouse had been developed and we called that mouse Emu-myc because this expresses
a particular oncogene and this mouse had been characterized and it's very tumor prone and will develop this B-cell lymphoma. So what we were able
to do, then, is to cross this with
the telomerase-null mouse and ask what happens when
telomerase is not there. Can that tumor go on and develop? And so of course, we have to be able to understand what happens when you first get rid of telomerase. So we look at the the first generation, the Emu-myc mice at are G1 and then of course, have to
breed and get the G2 mice, the G3 mice, et cetera, all the way up through the G6 mice to understand the role
of the short telomeres in this development of th
e tumor. So what we find is that there's a change in the survival of the
mice due to these tumors. And so I'll just walk you
through this graph here. This shows the percent
of mice that are alive at the beginning of the experiment versus the number of days. And so what's shown in the black line here is this mouse which has been made prone to these B-cell lymphomas. And so at the start, 100%
of the mice are alive but if you follow this black line, you can see that as time progresses, at 100 days,
about half
of these mice have died and they've all died of
this B-cell lymphoma. So they get this tumor
at a very high rate. If we now look at the mice
which were absent for telomeres, so they now don't have telomeres, but they're the very first
generation that lacked telomeres. What you find is a very
similar curve here in blue. So all of these mice are
dying of B-cell lymphoma and they're all have
succumbed to this lymphoma by about 200 days. However, in contrast, when we breed that out
to th
e sixth generation, where now the telomeres are
very short in these mice, what we find is that
although there are some mice that die early on, most
of the mice now survive and what you can see in these mice is that lymphoma started to grow but then it stopped growing
and it stopped growing because the telomeres were very short. And as I told you, the short telomeres then cause either cell death
or cellular senescence, so they couldn't go on
and form that tumor cell. And so this is one indication
that the the short telomeres will limit the growth of cells and in this setting, it's limiting the growth
of these cancer cells. So the short telomeres will
block the tumor growth. They don't block the early
stages that cause the cancer but what they do is they block the growth of the cancer cells. So the telomerase itself
isn't playing a role in the initiation of the cancer. However, when you have short telomeres, after those cells start
to grow for a while, you now block at this stage and the
cells can no longer go on and
form the full-blown tumor. So this was the work of David Feldser who was another graduate student in the laboratory who was breeding these telomeres-null mice
to the B-cell lymphoma mice. So the other aspect I would like to focus on now for the rest of the talk is the role of telomerase
in tissue renewal. So we were very interested
in some of the early effects that we saw in our 6th generation telomerase knockout mice. And we wanted to understand a little bit more
clearly what
happens in normal tissues when you don't have telomerase and the telomeres become short. So a number of years earlier, we had been involved in a collaboration with Cal Harley where we had shown and others have also
shown that if you take normal human cells and
you put those into culture and you grow them for a
number of cell divisions, it turns out what happens is that the telomeres
shorten progressively as the cells are dividing. So this is this end replication problem that I told
you about. And so what is shown
here is a representation of the distribution of telomere
lengths in human cells. Again, there's this heterogeneity of the telomere lengths. And as these cells are dividing, the length of this
telomere actually decreases as the cells are dividing in culture. So this was a first
indication that telomerase may play a role, that short telomeres may play a role in the inability of human cells to continue to divide. And then a similar
phenomenon is actually seen in peop
le, not in cells and culture, but rather with people of different age. So if you look at the mean telomere length and the way that we get
that mean telomere length, this is a representation
of the telomere length and so you can determine
how long a telomere is here, versus how long the mean telomere is here and if you simply plot
this on this graph here and look at mean telomere length versus the age of the person from which you took those blood cells, so this is now in normal
human white blood
cells, what you find is that there
is progressive shortening of the telomeres with increasing
numbers of cell division. Now, at first what we thought
that this might represent is the fact that these white blood cells might not have telomerase activity and therefore the telomeres
are shortening progressively. Although what I'm going to show you is that they do have
some telomerase activity but the number of times white blood cells have to divide outstrips the ability of the telomerase to be able
to keep up and elongate the telomeres. So over this long course
of cell divisions, what you see is this progressive telomere shortening in the bulk of the white blood cells
in all normal individuals. So the point that I'm going to make is that telomerase is limiting in cells. There's really just
enough telomerase activity in order to keep up with telomeres on the chromosomes that are where the telomeres are the shortest. So this story comes from a
number of different angles but the first hint th
at
the short telomeres might play a role in human disease came actually, again, from understanding molecular components
just like understanding the molecular component of
the telomere DNA sequence allowed us to understand what happens with telomere shortening. In this case, it's
understanding the structure of the RNA component. So the telomerase RNA has this structure here and it was first identified
with Cathy Collin's lab, here at Berkeley and also in my lab, that there's this
particular struc
ture here which binds to a set of proteins, in addition to this TERT protein which is necessary for
telomerase activity. There's a set of proteins
that binds to this structure and one of those proteins involves this protein
here called dyskerin. And this dyskerin protein was known to be involved in human genetic disease. And so that then led to just
the next year after this, a publication by a group
of human geneticists who were very interested in this disease, dyskeratosis congenita. And I'll t
ell you just in a second what dyskeratosis congenita is, but what was happening
here is that this group was very interested in this
inherited human disease. And so they were mapping, in families, this disease, not knowing what caused it. And by doing this genetic mapping, they were then able to
identify that in this case, the cause was the RNA
component of telomerase. So the title of the paper
that was published in 2001, the RNA component of telomerase is mutated in autosomal
dominant dyskeratos
is congenita. So this really galvanized people to understand how it is that telomerase can play a role in this
inherited genetic disease. So the clinical manifestations
of dyskeratosis congenita. It gets its name because
of skin and nail problems. Fingernails and toenails. That's why the term is dyskeratosis, because of the keratosis in the fingernails and the skin. So that's how the physicians
would see their patient, that was the most common visual manifestation of this disease. Although it wa
s known that
the mortality of the disease was due to bone marrow failure. Okay, so the inability of the bone marrow to continue making all
of normal blood cells that we require every single day. So this is an example of this requirement for stem cells to differentiate into all of the different
blood cell types. So that was the initial characterization of the human disease and the question was, how is it that the telomerase can play a role in this human disease? So we got involved with
this at Jo
hns Hopkins because a patient came in
to the hematology clinic at Johns Hopkins and as I was mentioning, we don't do science in a vacuum. We talked to our colleagues
and I was talking with a variety of the people
in the clinical departments to understand how to study
the blood cell effects and this patient came in, and this is the family of this patient. But this patient came into the clinic and was diagnosed as having this disease, dyskeratosis congenita. And so at the time, Mary Armanios decid
ed to understand
more about this family because what was known was
that this keratosis congenita was often mutations in this
RNA component of telomerase. So she brought these individuals within this family into the clinic and was able to identify
that there weren't mutations in the RNA component of
telomerase but there are mutations in the protein component of telomerase. So again, this is a
telomerase-mediated disease. And I show you this family, this individual that came into the clinic and hi
s father and his grandmother and those individuals here shown in black had these changes, these mutations, in the telomerase, in the family. And so this inheritance of this disease was shown to be autosomal dominant and I'll tell you a little
bit about what we know about that autosomal dominant inheritance. One of the remarkable
things about this family was that the the disease showed something that we call genetic anticipation. And that's a fancy way of saying that with each generation
that has
the disease, there's an earlier onset of the disease and more severity of the disease, okay? And this may remind you of
our telomerase knockout mouse. In the telomerase knockout mouse, we didn't see anything in
the first few generations but then we saw effects
in the later generations. And so the same thing
seemed to be occurring in this human family with
this inherited disease. The other thing that was
really quite remarkable is that those people that
were affected with the disease and had thi
s bone marrow failure actually were heterozygous. That is, they contained one normal copy and one mutant copy of
the telomerase gene. And that was quite remarkable to us because in a lot of
human genetic diseases, you have to have two mutant copies in order to actually have an effect. So this really got us thinking about how could it be that
you could be heterozygous, that is, have one normal
copy and one mutant copy. And so as geneticist,
what we thought about, was two molecular mechanisms
that
might underlie this inheritance pattern
in these families. And one of those is what's
called dominant negative and the other is haploinsufficiency. So what that basically means is that if you have one mutant
copy and one normal copy it could be that the
mutant copy actually binds to the normal copy and
takes it out of function. So that's a dominant mechanism. The mutant is dominant
over the normal copy. An alternative would be that
you have one mutant copy and one normal copy and
it's just not
enough to have one normal copy of the telomerase. And so we realized that
we were in the position where we could test this
genetically, directly in our mice. So that's what we set out to do, was to determine whether
there's a dominant effect or whether it's haploinsufficiency. And what I'll tell you, is our evidence that it's actually haploinsufficiency. It's just not enough to have only one copy of the functional telomerase. So what's shown here is a representation of the the telomeres again. T
his is a southern blot. But now we're looking at mice, so these are the the mice that we had initially been characterizing. And you can see that there's this very, very long, heterogeneous size to the telomeres in this
particular strain of mice. However, we looked at a variety of other strains of mice and
this is one that's shown here and you can see that the long telomeres that are here in this initial strain, now are represented by a
much less heterogeneous and shorter telomere
length in this
other strain of mice that we call castaneous, for Cast/Ei. And so we thought that
we would be able to see what's actually happening to telomeres much more easily on this
new genetic background. And I'll just represent that here. So if you imagine that you
have this telomere length with the signal intensity
of those red spots, the original mouse that we were using had very long heterozygous telomeres. However, this new mouse has much shorter, much more homogeneous telomeres. And so what we imagin
ed might happen, would be, after you have
telomere shortening, if there's a threshold
below which the telomeres have to go before you have some sort of functional consequence, when you have telomere
shortening in these settings, a much higher proportion of the cells would fall below this threshold when you have this much more
homogeneous telomere length. And so that's what we had set out to do, was to generate mice that now have a much more homogeneous
set of telomeres. And these experiments
act
ually were initiated by Mike Hemann, who I showed you earlier, and then continued by Ling-Yang Hao, who then really had some
remarkable discoveries. So what Ling-Yang set out to do, was to take these mice now, where we can see phenotypes more easily and to ask, what happens if these mice are always have one normal
copy and one mutant copy? That is, they are maintained
as heterozygous mice. Okay, so rather than breeding these mice and looking at the mice
that have no telomerase. Now, we're breedi
ng these mice and looking, just like
in the human studies, of always having the case
of having one normal copy and one wild-type copy. So that's what we call the
heterozygous generation one, heterozygous generation two, heterozygous generation three. And what we saw was, remarkably, there is progressive telomere shortening as you breed these mice
that have just one copy of telomerase instead of having
two copies of telomerase. And so I'll just quickly
show you that data, to make that point. So i
f you have a signal intensity here, and wild-type is shown in black so you have this telomere
length distribution. The knockout mouse have a shorter telomere length distribution, as
we had previously shown. But what was really important is that the heterozygous
generation one mice, shown in gray, are
shorter than the wild-type and more importantly, the
heterozygous generation five mice are shorter than the
heterozygous generation one, showing that we actually have progressive telomere shortening
when
you have haploinsufficiency. So this was quite remarkable. And that gets back to the point again, that telomerase is limiting in cells. That having just half the level over a number of generations
doesn't allow you to be able to maintain telomere length. If you now look at the
survival of those mice and this is, once again,
one of those survival curves that I showed you earlier with the percent of mice alive
versus the number of days and I'll just have you focus
on two of these curves. The
wild-type, so in black up here, is the wild-type mice over 400 days. And down here are the
heterozygous generation 10 mice. Now, you see that there's
a significant death of these mice due to the short telomeres. And there's actually,
with each generation, there's a progressive decline
in the viability of the mice. And this looks very much
like the genetic anticipation that's seen in the human families that have dyskeratosis congenita. So the inheritance of short
telomeres decreases survival. An
d as I said, this resembles the genetic anticipation seen
in dyskeratosis congenita. So these telomerase deficient mice have many of the same defects that are seen in the human patients. Bone marrow failure, intestinal atrophy, immune senescence, they're
chemotherapy intolerant and they show this genetic anticipation. And the mortality in these mice is due primarily to these
immune system defects. And all of these experiments
would not be possible, were it not for a really remarkable mouse handl
er here, Margaret Strong, who did all of the intricate
breedings for these mice. So one of the most interesting things about this set of experiments was of course, every time you breed two mice that are heterozygous, have one copy that's
wild-type and one mutant, you get out a heterozygous generation and we can breed these
for many generations but at each stage of breeding, we get out, of course, the wild-type, the next generation heterozygote, and we get out the knockout. And I can remember whe
n Ling-Yang Hao came into my office one day and he said, I wonder what would happen
if we looked at the telomeres of the wild-type mice that
come from these crosses. And so he did that and
what I'll show you here, is we designate that wild-type star because it turns out that
with the progressive breeding in the heterozygous state,
even the wild-type telomeres that come out were
short because telomerase is limiting and the telomere
lengths weren't restored. So that's shown here. Again, here's the
intensity of the telomeres, the length of the telomeres. Here's the wild-type telomere length, the knock out just for reference. The heterozygous generation
five I showed you earlier is shorter than wild-type
but quite remarkably, the wild-type five star,
that is the the littermates, the brothers and sisters
from these heterozygous mice also have telomeres that are significantly shorter than their wild-type counterparts. And that's because telomerase is limiting. You don't have enough
telomeras
e to go around and top up all of the telomeres
in just one generation. So I've been making the point to you that it's the short telomeres that are causing these phenotypes. When the telomeres get short, you cause a damage
response and the cells die. So quite remarkably, when
we looked in these mice, these wild-type star mice
also had the same effects as the mutant mice had. And this, as I said, is quite remarkable. So if we look at either
the testis apoptosis, that is the the cell death
that occ
urs in the testes or the immune system defects, if we look at the spleen weight, the wild-type star mice
had significant reductions. So this was really quite remarkable because now we have an
inherited genetic defect but even now in the case where
the genetics is restored, the telomere length hasn't been restored and we still have an effect. So we called this genetic disease in the absence of the telomerase mutation that caused the disease, we called that an occult genetic effect, a hidden genet
ic effect. And it's because the telomeres themselves haven't yet been restored to
their normal telomere length. And now, this might be a kind of a cute little trick that is, okay, so we have something, we
now call it occult genetics, but it actually matters because recall, this is an inherited human genetic disease and you have these
individuals walking around that have heterozygous mutations and the only way to treat a
number of these individuals, for instance, who have
bone marrow failure, is
with a bone marrow transplant. So you can imagine, if you have telomerase
mutation in the family and you have one brother that needs a bone marrow transplant
because he has short telomeres, that his brother or his cousin, who may not have the
mutation in telomerase, might still have short telomeres. And so it's important to
know what the telomere length is of the the donor bone marrow rather than just the genetics. So this actually matters to the treatment of this human disease. So it's the shor
t telomeres, not the absence of telomerase
that's causing the disease. So just to round out this story, it turns out that having identified this genetic anticipation where you see this worsening of
phenotype and earlier onset of this bone marrow failure, it turns out that there are other diseases that these short telomeres are causing besides the bone marrow failure. And so the first one to be identified was something called
idiopathic pulmonary fibrosis. Idiopathic because we
didn't know what c
aused it and it turns out that it's
caused by the short telomeres. And this was recognized by Mary Armanios because she saw this genetic anticipation in this family that had
come into the clinic. And so it was identified that it was actually telomerase mutations that underlie this other
human genetic disease. And as we've been able
to understand more fully, Mary has been able to characterize what she calls a syndrome
of telomere shortening. That is, there are a
number of different effects of hav
ing these short telomeres. There are these bone marrow
failure effects in the blood, the lung effects, the pulmonary fibrosis, liver disease, and as these studies are continuing and ongoing, it's clear that there are going to be a
number of different effects. And very interestingly,
all of these diseases are actually diseases that are associated with the normal aging process. That is, they are normal,
age-related degenerative disease. So even in families
that haven't necessarily inherited one of
these mutations, if you have short telomeres,
you may be at risk for a number of these age-related
degenerative diseases, due to the short telomeres. So this is taken from another group and this is just an example of what I had shown you previously. If you look at telomere length versus age and this is now just
looking at white blood cells from normal people, what you see is that there is this decrease
in telomere length with age but also what's really quite remarkable is there's a huge amount
of heterogeneity within the human population. If you look at a large
number of individual people, you find that there's this great decrease and there's been a
little bit of information in the lay press about how, maybe, telomeres can tell you how old you are. However, due to this, of course, hopefully you would know how old you are. But (laughs) the telomeres could determine, if you didn't
know how old someone was. But if you look at the heterogeneity here and I gave you a sample of DNA and that
DNA had telomeres that were 7kb. Okay, that person could be 20 or that person could be 70. So it's not something
that's so determinative that we can say for sure
if you have a certain telomere length that
there's a particular effect. However, this heterogeneity and this decrease in telomere
length in the population, those individuals that have
the shortest telomeres, we know are at risk for disease. So you can model this
heterogeneity in the population with confidence intervals
and this was don
e by Peter Lansdorp and Mary Armanios has also done that at Johns Hopkins. And so what you get here is
this normal distribution. So if you look at telomere
length versus age, these lines here were developed
on 400 normal individuals and you can say that if
you're above this line, then that's the 99th
percentile of telomere length. So this is the normal distribution of telomere length in the population. And if you look, now, in a
number of these families, where you see this
inherited genetic dise
ase, those people in the families that do not have the telomerase mutation fall here around this 50th percentile. However, those individuals
that do have the mutation typically fall below the first percentile. So those individuals with
the shortest telomeres are the ones that are at risk
for the bone marrow failure, the pulmonary fibrosis, and
the liver complications. And as I hope that I've
been making apparent, that since it's the short telomeres, not the mutation itself
that causes the diseas
e, any individual that falls
below the first percentile may be at risk for these disease, not just those that are in these families with these inherited syndromes. So many diseases that are seen in families with insufficient
telomerase share features of these age-related degenerative disease. So we know that bone marrow failure is, in the normal population,
an age-related complication. As is immune senescence,
chemotherapy intolerance. Pulmonary fibrosis typically is diagnosed in the sixth or se
venth decade. Liver disease and also, paradoxically, there's increased cancer incidence. So the short telomeres may play a role in the wider population,
not simply in the subset of people with inherited
mutations in telomerase. So just to summarize, I've told you that telomeres are required for
chromosome end protection. Telomerase is essential for
telomere length maintenance. Telomere shortening leads
to either cell death or cell senescence after
many cell divisions. Short telomeres limit tissu
e renewal and contribute to age-related
degenerative disease. And then, just to come back to the point about fundamental discoveries, new discoveries often
come from unlikely places. When we set out to understand how it is that telomeres are maintained on the ends of chromosomes,
due to replication, we weren't looking to understand age-related degenerative disease or cancer but by following our curiosity, it turns out that the
telomere clearly plays a role in both these diseases. So curiosity-dr
iven research provides unexpected discoveries
that can have important implications for human health. And these are the current
members of my laboratory. We collaborate with Mary Armanios and her group and many of the experiments that I told you were done by past members. And this is just a slightly older picture of my laboratory when they
all dressed up as chromosomes. (audience laughing) And then the laboratory currently, it's a little out of focus but this was the political campaign for the Am
erican Society of Cell Biology. We are research giving a face to basic science. So I'll stop there. (audience applauding) (upbeat music)
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